RNA polymerase II (Pol II) plays a critical role in readout of the eukaryotic genome through its transcription of protein-coding and non-coding RNAs. Normal physiological processes depend upon precise transcriptional controls, whereas transcriptional dysregulation is the basis of numerous pathologies that include cancer. Pol II recruitment to specific promoters is regulated by multiple (co)factors that include the multi-subunit Mediator, which directly binds both Pol II and transcriptional activators to facilitate activation of essentially all genes. Pol II(G) is a ditinct, recently discovered (originally in calf thymus and pig liver) form of Pol II that contains the tighly associated polypeptide Gdown1 along with the normal 12 subunits of Pol II. In vitro transcription studies revealed that, in the absence of Mediator, the Pol II-associated Gdown1 represses transcription by preventing the association of initiation factor TFIIF with Pol II and thus inhibitng pre-initiation complex formation. Mediator reverses the repressed transcriptional capacity of Pol II(G), although the underlying mechanism remains unclear. While Mediator is conserved from yeast to human, the conservation of Gdown1 extends only to Drosophila melanogaster. Thus, the transcriptional regulatory system in fruit flies and higher metazoans has evolved through acquisition of an additional and functionally distinct Pol II, Pol II(G), although the specific rol(s) played by Pol II(G) in transcriptional regulation in higher metazoans remains unclear. Toward an understanding of the biological role(s) of Pol II(G), preliminary studies have demonstrated that Gdown1 ablation causes early embryonic lethality in both fruit flies and mice. This lethality may be explained in part by a severe inhibition of cell proliferation, as observed in Gdown1-deficient embryonic stem cell and cancer cell lines, and by dysregulation of gene expression, as observed in Gdown1-deficient hepatocytes. However, it seems unlikely that Pol II(G) is important primarily for cell growth since Gdown1 is not conserved in yeast and lower metazoans. With the general objective of understanding the in vivo roles and Mediator-dependent activation mechanisms for Pol II(G), our specific aims are: (i) to investigate the biological functions of Pol II(G) in embryonic and adult cells through determination of genes directly regulated by Pol II(G) -- by RNA-seq and ChIP-seq assays with Gdown1 null blastocysts, conditional Gdown1 knockout mouse, mouse embryonic stem cells, and Gdown1-null hepatocytes); (ii) to investigate the molecular mechanism of Mediator-dependent Pol II(G) transcription by genetic and biochemical determination of contact sites between Pol II, Gdown1 (wt and mutant) and Mediator and by CX-MS and cryoEM structural analyses of corresponding complexes; (iii) to examine the impact of Gdown1 ablation on tumorigenesis using mouse models that involve xenograft of human cancer cells and hydrodynamic injection of oncogenic vectors to generate liver cancer. Thus, in addition to revealing biological roles of Pol II(G) and the mechanism underlying its regulation by Mediator, these studies may reveal new targets for cancer therapeutics.